Methods for screening drugs to predict tardive dyskinesia

ABSTRACT

The present invention provides screening methods for identifying compounds which induce tardive dyskinesia (TD) when administered to an animal. In particular, the methods involve assaying for intermediates and end product of reactions associated with candidate compound mediated reduction of reducible substrates. Also provided are high-throughput screening methods for determining whether compounds induce TD when administered to an animal. Further, provided are methods for treating psychoses comprising testing antipsychotic drugs to identify those which will not induce TD when administered to an animal and administering one or more such drugs to a patient in need thereof.

[0001] This application claims the benefit of the filing date ofprovisional application 60/060,962, filed on Oct. 6,1997, which isherein incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention provides a screening method for identifyingcompounds which induce tardive dyskinesia (TD) when administered to ananimal.

[0004] 2. Related Art

[0005] Tardive dyskinesia (TD) is a debilitating side effect oflong-term antipsychotic exposure. This movement disorder affects 20-40%or more of patients treated chronically with antipsychotic drugs(Morgenstern, H. and Glazer, W. M., et al., Arch. Gen. Psychiat.50:723-733 (1993)) and roughly 4-5% of patients are expected to developTD with each year of antipsychotic treatment. The manifestations of TDmay include adventitious movements of the oral-facial region,choreoathetosis of extremities and lordotic posturing. Despite therecent arrival of atypical antipsychotic such as clozapine andolanzapine, large numbers of patients continue to receive conventionalantipsychotics.

[0006] The pathophysiologic basis for antipsychotic-induced TD remainsunclear. While the theory that striatal postsynaptic dopamine receptorsupersensitivity causes TD has been widely accepted for two decades,some evidence contradicts this model (Jenner, P. and Marsden, C. D.,TINS 9:259-260 (1986)). Although acute administration of antipsychoticstemporarily increases the firing of dopamine neurons, chronicantipsychotic treatment leads to a decrease in their firing rate due todepolarization blockage (Cadet, J. L. and Lohr, J. B., Ann. N.Y. Aca.Sci. 570:176-185 (1989)). After an acute elevation of dopamine turnoverwith haloperidol treatment, dopamine synthesis and release decreases;and dopamine metabolite levels return to normal with chronic treatment(Rastogi, S. K., et al., Prog. Neuro-Psychopharmacol. & BioL Psychiat.7:153-164 (1983)). Notably, 5 striatal dopamine metabolites are reducedin monkeys with dyskinesia due to long-term antipsychotic treatment.Finally, post mortem neurochemical studies have not revealed acorrelation between dopamine receptor up-regulation and TD (Crow, T. J.,et al., Journal of Clinical Psychopharmacology 2:336-340 (1982). Thus,excessive activation of postsynaptic striatal dopamine receptors is notconsistent with the time course nor persistence of TD.

[0007] An alternative hypothesis supports a neurodegenerative processaffecting striatal efferent neurons analogous to Huntington's diseaseand Wilson's disease.

[0008] TD is similar to these diseases in that all three diseasespresent choreoathetoid movements and pathological changes in thestriatum. Christensen et al. (Christensen, E., et al., Acta PsychiatricaScandinavica 46:14-23 (1970)) reported neuronal loss in the basalganglia of patients with persistent TD; similar losses have beendescribed in rats treated chronically with antipsychotics (Pakkenberg,H., et al., Psychopharmacologia 29:329-336 (1973); Nielsen, E. B. andLyon, M., Psychopharmacology 59:85-89 (1978); Gunne, L. M. and Andren,P. E., Clin. Neuropharmacol. 16:90-95 (1993)). Importantly, loss of thepresynaptic markers for the striatal-pallidal and nigral GABAergicneurons, glutamic acid decarboxylase (GAD) and γ-aminobutyric acid(GABA), have been observed in a primate model for TD and in post mortemstudies of patients with TD (Gunne, L. M., et al., Nature 309:347-349(1984); Gunne, L. M. and Haggstrom, J. E., Journal of ClinicalPsychiatry 46:48-50 (1985); Anderson, U., et al., Movement Dis. 4:37-46(1989)).

[0009] Both preclinical and clinical studies point to degeneration ofstriatal efferent neurons, especially GABAergic neurons, in TD. Althoughinconclusive, one brain imaging study has revealed reduction in thevolume of the caudate nuclei in patients with TD in comparison topatients without TD and normal controls (Mion, C. C., et al., PsychiatryResearch 40:157-166 (1991)). In the primate model of experimental TD,antipsychotic induced dyskinetic monkeys exhibit a reduction inpresynaptic GABAergic markers in the subthalamic nucleus, the medialsegment of globus pallidus and rostral part of the substantia nigra(Gunne, L. M., et al., Nature 309:347-349 (1984)). Rodent models of TDhave also revealed a significantly lower density of large neurons in thestriatum (Jeste, D. V., et al., Psychopharmacology 106:154-160 (1992)),and decreased GAD activity in the substantia nigra (Gunne, L. M. andHaggstrom, J. E., Journal of Clinical Psychiatry 46:48-50 (1985); Jeste,D. V., et al., Psychopharmacology 106:154-160 (1992)). Finally, Mitchellet al. have recently demonstrated rat apoptotic neuronal death in thestriatum as a consequence of removal of the nigrostriatal dopaminergicpathway, similar to antipsychotic drug induced dopamine antagonism(Mitchell, I. J., et al., Neuroscience 63:1-5 (1994)).

[0010] We have found elevated levels of CSF N-acetylaspartate (NAA) inTD patients. This finding is consistent with a neuronal degenerativeprocess since CSF NAA, a marker for neuronal integrity, is elevated inamyotrophic lateral sclerosis (Rothstein, J. D., et al., Ann. Neurol.28:18-25 (1990)); and tissue NAA levels decrease in areas involved inactive neuronal degeneration in amyotrophic lateral sclerosis,Huntington's Disease and Alzheimer's Disease (for a review, Tsai, G. andCoyle, J. T., Prog. in Neurobiol. 56:531-540 (1995)).

[0011] The incidence rate of TD increases throughout the patient'sexposure to antipsychotic drugs. The longer the exposure, the higher thepatient's risk of developing TD. This phenomenon points to theinadequacy of preclinical trials which only assess the risk of TD duringthe trial period. This phenomenon also suggests that the neuronalinsults associated with TD are cumulative and the process which leads tothis disease may be insidious and subtle in nature.

[0012] There is accumulating evidence suggesting that antipsychotics caninduce oxidative stress through a variety of mechanisms. Elevated levelsof conjugated dienes and thiobarbituric acid reactive products (TBARS)in the CSF of TD patients have been reported (Pall, H. S., et al.,Lancet ii:596-599 (1987); Lohr, J. B., et al., Biol. Psychiat.28:535-539 (1990); Jeste, D. V., et al., Psychopharmacology 106:154-160(1992)).

[0013] Elevated oxyradicals can inhibit presynaptic glutamate uptake,inactivate the enzymatic defenses against cellular oxidants (Volterra,A., et al., J. Neuroscience 14:2924-2932(1994)) and disruptmitochondrial electron transport (Burkhardt, C., et al., Ann. Neurol.33:512-517 (1993); Jackson-Lewis, V. and Przedborski, S., Ann. Neurol.35:244-245 (1994)), which results in an increased generation ofsuperoxide and extraneuronal excitatory amino acids. Persistentactivation of glutamate ionotropic receptors has long been known tocause neuronal degeneration (Olney, J. W., Annu. Rev. Pharmacol.Toxicol. 30:47-41 (1990)). Recent studies indicate that oxidative damagemediates the delayed neuronal degeneration caused by activationofN-methyl-D-aspartate NMDA) and non-NMDA glutamate ionotropic receptors(Coyle, J. T. and Puttfarcken, P., Science 262:689-695 (1993)). Thisoxidative damage can be gradual, insidious and cumulative leading to anapoptotic form of neuronal death. It provides an important pathologiclink between moderate levels of excessive glutamate ionotropic receptorstimulation and delayed neuronal degeneration.

[0014] The sources of oxyradicals produced as a consequence of long-termantipsychotic exposure are diverse and remain incompletelycharacterized. Depolarization activates oxidative metabolism of glucosevia the mitochondrial electron transport chain with the superoxideradical generated as a byproduct. Recent reports indicate thatantipsychotics inhibit the mitochondrial electron transport chain atComplex I, which would further enhance superoxide generation (Burkhardt,C., et al., Ann. Neurol. 33:512-517 (1993); Jackson-Lewis, V. andPrzedborski, S., Ann. Neurol. 35:244-245 (1994)), and CSF metabolicabnormalities found in this cohort of patients is consistent with animpairment of mitochondrial electron transport at Complex I (Goff, D.C., et al., Amer. J. Psychiat. 152:1730-1736 (1995)). Anotherpossibility, however, is that an increased turnover of catecholaminesresults in free-radical formation. Nevertheless, it is unclear whetherantipsychotics can directly generate free radicals and a reliablescreening method for the potential of antipsychotic-induced TD does notexist.

[0015] The hypothesis of oxidative damage-to striatal neurons mediatedby antipsychotic enhancement of glutamatergic neurotransmission issupported by recent reports that Vitamin E reverses the symptoms of TD.The anecdotal reports have been sustained by double blind placebocontrolled studies with Vitamin E (Egan, M. F., et al., Amer. JPsychiat. 149:773-777 (1992); Adler, L. A., et al., Amer. J Psychiat.150:1405-1407 (1993)). Notably, those patients earlier in the course oftheir disorder are more responsive to treatment with Vitamin E,consistent with the model that the oxidative damage is cumulative overtime and involves functional impairment prior to frank degeneration.Similarly, in a double blind placebo controlled study of Vitamin Etreatment of patients with Huntington's Disease, those who were lesssymptomatic at the initiation of treatment exhibited the most favorableresponse (Peyser, C. E., et al, Amer. J Psychiatry 152:1771-1775(1995)). Inasmuch as centrally active free radical scavengers may notonly reverse the oxidative damage, additional studies on oxidativestress in TD as well as efficacy of prevention and treatment withcentrally active free radical scavengers on the surrogates oxidativestress in TD need to be carried out.

[0016] One of the major sources of potential oxidative stress in thebrain is redox active metals (reviewed in Markesbery, W. R., Free Radic.Biol. Med. 23:134-147 (1997)). Iron and copper are highly concentratedin the basal ganglia. Reduction of iron (III) and copper (II) generatesiron (II) and copper (I), respectively. In Wilson's disease, there isprogressive accumulation of copper within the body tissues, particularlythe erythrocytes, kidney, liver and brain. In the blood, more than 90%is found in the plasma associated with ceruloplasmin. Copper absorptionappears to be accelerated, and although the urinary excretion of freecopper is usually increased, affected individuals of Wilson's diseaseare in positive copper balance. In addition to Wilson's disease,abnormal copper metabolism exists in the neurodegenerative disorders ofMenkes' syndrome and possibly familial amyotrophic lateral sclerosis.Caudate, putamen, cerebral cortex and the dentate nuclei are thevulnerable regions in Wilson's disease. When cerebral copperaccumulation is sufficient to destroy the nerve cells, the neurologicalsyndrome begins. The most common features of this disease includechoreoathetoid movements, muscular rigidity, and tremor of theextremities which have remarkable similarity to the symptoms of TD.

[0017] Although the relevance of oxidative stress to TD is becomingclear, a rational neurochemical basis for developing antipsychoticsdevoid of TD is lacking. While Vitamin E treatment has been used withsome success, drugs aiming at the source of oxidative stress in TD ismissing. The application of other antioxidants will not address theunderlying pathogenesis mechanism.

SUMMARY OF THE INVENTION

[0018] The present invention provides screening methods for identifyingcompounds which induce TD when administered to an animal. These methodsinvolve contacting a candidate compound with one or more reduciblesubstrates, assaying for the production of one or more reactionintermediates or products associated with the reduction of the reduciblesubstrate(s), and comparing the production of reaction intermediate(s)or product(s) to a standard production of reaction intermediate(s) orproduct(s), the standard being determined using a standard reactionmixture which does not contain the candidate compound. The production ofsignificant quantities of reaction intermediate(s) or product(s) overthe standard indicates that the candidate compound induces TD whenadministered to an animal.

[0019] Aspects of the present invention include screening methods foridentifying compounds which induce TD when administered to an animal,wherein the ability of the candidate compound to (a) reduce a reduciblesubstrate from a first oxidation state to a second lower oxidation stateor (b) engage in chemical reactions which result in the production ofone or more free radicals, precursors or intermediates of free radicals,or reactive oxygen species is measured. As above, the ability of thecandidate compound to reduce the reducible substrate or engage in thespecified chemical reaction(s) is determined by comparison to a standarddetermined in the absence of candidate compound.

[0020] Preferred methods of the present invention comprise assays whichmeasure the ability of a candidate compound to:

[0021] (a) reduce a reducible substrate (e.g., copper (II) or iron(III)) from a first oxidation state to a second lower oxidation state;

[0022] (b) engage in chemical reactions which result in the productionof peroxides (e.g., hydrogen peroxide); or

[0023] (c) engage in chemical reactions which result in the productionof free radicals (e.g., hydroxyl radicals).

[0024] The present invention also provides methods employingcombinations of more than one assay of the present invention, wherein asignificant deviation from the standard reaction mixture in each ofthese assays confirms that the candidate compound induces TD whenadministered to an animal.

[0025] The present invention also provides combinations of one or moreof the assays of the present invention and testing of the candidatecompound in an animal model (e.g., a rodent model) to confirm that thecandidate compound induces TD when administered to an animal.

[0026] The present invention further provides combinations of one ormore of the assays of the present invention and testing of the candidatecompound for additional activities (e.g., superoxide dismutase activity,catalase activity) to determine whether the candidate compound inducesTD when administered to an animal.

[0027] The present invention also provides high-throughput assays forscreening candidate compounds to identify those compounds which induceTD when administered to an animal.

[0028] The present invention further provides methods for treatingpsychoses comprising the testing of candidate compounds to identifythose which will not induce tardive dyskinesia when administered to ananimal, and administering one or more of these compounds to a patient inneed thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0029]FIG. 1 shows the results of copper and iron reduction assay forvarious antipsychotic drugs. The data were generated using the methodsdisclosed in Example 1(a).

[0030]FIG. 2 shows the results of copper and iron reduction assays forvarious non-antipsychotic psychotropic drugs. The data were generatedusing the methods disclosed in Example 1(a).

[0031]FIG. 3 shows the results of hydrogen peroxide production assaysfor various antipsychotic drugs. The data were generated using themethods disclosed in Example 1(b).

[0032]FIG. 4 shows the results of hydrogen peroxide production assaysfor various non-antipsychotics. The data were generated using themethods disclosed in Example 1(b).

[0033]FIG. 5 shows the dose response of the generation of hydrogenperoxide by fluphenazine. The data were generated using the methodsdisclosed in Example 1(b).

[0034]FIG. 6 shows the relationship between copper concentration and thegeneration of hydrogen peroxide production by Vitamin C and theantipsychotic drugs chlorpromazine and haloperidol. The data weregenerated using the methods disclosed in Example 1(b).

[0035]FIG. 7 shows that the chelating agents bathocuproine disulfonate(BC) and triethylenetetramine (TETA) significantly attenuate theproduction of hydrogen peroxide by Vitamin C and antipsychotics. Thedata were generated using the methods disclosed in Example 1(b).

[0036]FIG. 8 shows that oxygen augments and argon attenuates theproduction of hydrogen peroxide production by Vitamin C andantipsychotic drugs. The data were generated using the methods disclosedin Example 1(b).

[0037]FIG. 9 shows the production of hydroxyl radical by antipsychotics.The data were generated using the methods disclosed in Example 1(c).

[0038]FIG. 10 shows the production of hydroxyl radical bynon-antipsychotic psychotropic drugs. The data were generated using themethods disclosed in Example 1(c).

[0039]FIG. 11 shows the redox reactions predicted to occur in vivo whenantipsychotic drugs which induces TD are administered to an animal.

[0040]FIG. 12 shows vacuous chewing movement in rats during a 36 weektime course treated with fluphenazine (TD and non-TD groups), clozapineand vehicle for the first 24 weeks and withdrawn for the final 12 weeks.

DETAILED DESCRIPTION

[0041] Definitions

[0042] The following definitions are provided to clarify the subjectmatter which the inventors consider to be the present invention.

[0043] As used herein, the phrase “candidate compound” refers to bothantipsychotic drugs and compounds potentially useful as antipsychoticdrugs which are screened by the methods of the present invention todetermine whether these compounds induce TD. The term “compound”includes synthetic molecules and naturally occurring products, whetherpresent in purified form or crude mixture.

[0044] As used herein, the term “animal” refers to members of the animalkingdom. Preferred animals are mammals. Most preferred animals arehumans.

[0045] As used herein, the phrase “reactive oxygen species” refers tomolecules (and singlet oxygen) which contain oxygen and are generallyeither toxic to biological systems or readily engage in reactions whichproduce toxic by-products. Reactive oxygen species include superoxideanions (O₂ ^(•−)), hydroxyl radicals (OH^(•)), and hydrogen peroxide(H₂O₂). These reactive oxygen species do not include molecular oxygen(O₂).

[0046] As used herein, the phrase “reducible substrate” refers to anelement or complex ion which can be reduced in a redox reaction. Thestandard reduction potential of the redox reaction for reduciblesubstrates useful in the practice of the present invention willgenerally be below 2.0 E°/V.

[0047] As used herein, the phrase “complex ion”refers to an ion which isnot an element. Examples of complex ions include compounds such asMn(OH)₃, Pt(OH)₂, and proteins. Proteins are considered to be complexions, even if they do not have a net positive or negative charge, aslong as they have regions which can participate in redox reactions.

[0048] As used herein, the phrase “standard reaction mixture” refers toa control reaction mixture which contains all of the ingredients of areaction mixture used to assay for determining whether a candidatecompound can induce TD except for the candidate compound. This mixturemay be assayed before, at the same time, or after the reaction mixturecontaining the candidate compound. The standard reaction mixture is alsoreferred to herein as a “blank”.

[0049] As used herein, the phrase “tardive dyskinesia”, abbreviated“TD”, refers to an affliction characterized by involuntary movement ofmuscles resulting from neuronal degeneration induced by antipsychoticdrugs.

[0050] As used herein, the term “antipsychotic” and the phrase“antipsychotic drug” refer to psychotropic drugs used in the treatmentof psychoses. For purposes herein, the terms “antipsychotic” and“neuroleptic” are equivalents.

[0051] As used herein, the term “psychosis” (plural being “psychoses”)refers to a mental disorder which causes gross disorganization of aperson's mental capacity to recognize reality and to communicate andrelate to others to the degree that the disorder interferes with theafflicted person's ability to cope with the occurrences of everydaylife.

[0052] Additional definitions are provided throughout the specification.

[0053] Screening Methods of the Present Invention

[0054] We have found the cerebrospinal fluids of TD patients have lowerSOD activity and higher levels of protein carbonyl groups than TD-freesubjects. SOD is an enzyme critical in the detoxification of superoxide,a byproduct of oxidative metabolism. These results suggest thatattenuated activity of SOD may contribute to increased oxidation ofprotein. Chronic treatment of rats with fluphenazine has been reportedto cause a decrease in SOD and catalase activities in the nervoussystem. Decreased SOD renders neurons more vulnerable to oxyradicalinjury, consistent with the elevated levels of protein carbonyl groups.While we did not observe an elevation in lipid hydroperoxides in CSF inTD, elevated levels of conjugated dienes and thiobarbituric acidreactive products (TBARS) in the CSF of TD patients have been reported.These findings are consistent with the final results of oxidativedamage. Nevertheless, the pathophysiological basis ofantipsychotic-induced oxidative stress in unclear.

[0055] The purpose of the present invention is to investigate theunderlying pathophysiological mechanism of TD, which we hypothesize is acumulative process, and provide an in vitro tool for determining thepotential of antipsychotics to induce TD in vivo. Thus, the presentinvention provides a facile tool for the screening of candidatecompounds, such as candidate antipsychotic drugs, which predict thecompound's likelihood of inducing TD when administered to an animal.This is based upon our discoveries relating to the underlyingpathophysiological mechanism of TD, which we hypothesize is due to anoxidative stress-related cumulative process caused by the redoxchemistry antipsychotic drugs themselves. As shown in FIG. 11,antipsychotic drugs are believed to induce TD when administered to ananimal as a result of their ability to convert Cu(II) to Cu(I). Thisability to reduce copper ions in vivo is thought to result in theformation of reactive oxygen species which induce neuronal oxidativedamage. (FIG. 11.)

[0056] Metal ions are believed to engage in reactions which result inthe production of reactive oxygen species in biological organisms(reviewed in Markesbery, W. R., Free Radic. BioL Med. 23:134-147(1997)). One series of such reactions, shown below, concludes with theFenton reaction, shown in (c), and the formation of hydroxyl radicals.

[0057] (a) Reduced Compound+O₂→ Oxidized Compound+O₂ ^(•−)(e.g., Fe²⁺,Cu¹⁺) (e.g., Fe³⁺, Cu²⁺)

[0058] (b) O₂ ^(•−)+O₂ ^(•−)+2H⁺→H₂O₂+O₂

[0059] (c) Reduced Compound+H₂O₂ → Oxidized Compound+OH^(•)+OH⁻(e.g.,Fe²⁺, Cu¹⁺) (e.g., Fe³⁺, Cu²⁺)

[0060] As shown in FIG. 9, most of the antipsychotics currently in useand known to cause TD generate the hydroxyl radical (OH^(•)) by Fentonchemistry. Like Aβ amyloid, these antipsychotics have the capacity toreduce Cu(II) to Cu(I), and form hydrogen peroxide (H₂O₂) (FIGS. 1 and3, respectively) from the apparently spontaneous reduction of molecularoxygen to superoxide, simultaneously. The reduced Cu(I) and H₂O₂ reactto generate the hydroxyl radical. No other class of drug or psychotropicis capable of generating OH• in this manner, and clozapine, the onlyantipsychotic with a low potential for inducing TD as an adverse effect,is also unable to generate OH•.

[0061] Clozapine presents an exception to the correlation between copperreduction activity and the ability to induce TD by antipsychotic drugs.As shown in FIG. 1, clozapine has potent copper reduction activity whichsuggests that this drug induces TD when administered to an animal. Ourdata indicates, however, that this drug may exhibit superoxide dismutaseand catalase activities which apparently scavenge hydroxyl radicalprecursors. This conclusion is supported by the fact that, as shown inFIG. 9, clozapine does not generate significant amounts of hydroxylradicals.

[0062] We have adapted this system into a rapid through means fordetermining which compounds generate OH• and therefore predicting whichcompounds would be candidates for causing TD. Our invention provides asystemic appraisal of the TD-inducing risk of antipsychotics. From ourinvention, antipsychotic drugs can be identified which should not becapable of either reducing Cu(II) or generating reactive oxygen speciesin vivo upon administration to an animal. Thus, the present inventionfurther allows for the identification of compounds which will not induceTD when administered to an animal. Such compounds may be identified, forexample, by their inability to reduce a reducible substrate or engage inchemical reactions which result in the production of free radicals,precursors of free radicals, or reactive oxygen species in vitro.

[0063] The present invention can be practiced using virtually any methodwherein the ability of a candidate compound to reduce a reduciblesubstrate from a first oxidation state to a second lower oxidation stateor engage in chemical reactions which result in the production of freeradicals, precursors of free radicals, or reactive oxygen species ismeasured.

[0064] In each embodiment of the invention, the data obtained in thepresence of the candidate compound is compared to a standard reactionmixture which is essentially identical to that containing the candidatecompound except the candidate compound is absent. This standard reactionmixture may be assayed at the same time as the reaction mixturecontaining the candidate compound or may be assayed prior or subsequentto the mixture containing the candidate compound.

[0065] As noted above, the screening methods of the present inventionare based on the redox chemistry of the candidate compound itself whichis believed to be responsible for the induction of TD. Thus the presentinvention encompasses any assay method which identifies characteristicsof candidate compounds which result in the induction of TD whenadministered to an animal. As shown in FIG. 1, one characteristic ofcompounds which induce TD is the ability to reduce Cu(II) to Cu(I). Asalso shown in FIG. 1, these compounds also have some Fe(III) reductionactivity. The ability of antipsychotic drugs to reduce Cu(II) morereadily than Fe(III) is believed to be related to the standard reductionpotentials of each ion. As shown in Table 1, the standard reductionpotential for the conversion of Cu(II) to Cu(I) (0.153 E°/V) is muchlower than for the conversion of Fe(III) to Fe(II) (0.771 E°/V). Thus,the present invention encompasses methods for identifying compoundscapable of reducing ions with standard reduction potentials between 0and 2.0 E°/V, more preferably between 0 and 1.6, 0 and 0.8, 0 and 0.6,or 0 and 0.4 E°/V and most preferably between 0 and 0.2 E°/V. TABLE 1Representative Examples of Standard Reduction Potentials.* Reaction E°/VMn(OH)₃ + e

Mn(OH)₂ + OH⁻ 0.15 Cu²⁺

Cu⁺ 0.153 BiCl₄ ⁻ + 3e

Bi + 4 Cl⁻ 0.16 Fe³⁺

Fe²⁺ 0.771 Mn³⁺

Mn²⁺ 1.5415

[0066] The standard reduction potentials of several ions are shown inTable 1. Since, as noted above, the induction of TD by antipsychoticdrugs is linked to the compounds redox chemistry, one skilled in the artcould readily identify reducible substrates potentially suitable for usein the practice of the present invention. For example, as shown in Table1, the standard reduction potential for the conversion of Cu(II) toCu(I) (0.153 E°/V) is significantly lower that for the conversion ofFe(III) to Fe(II) (0.771 E°/V) and Cu(II) is more readily reduced thanFe(III) (FIG. 1). By using suitable assays, the inventors believe thatcandidate compound induced reduction of reducible substrates havingstandard reduction potentials as high as 2.0 E°/V may be quantified.

[0067] The present invention also encompass assays which measure thereduction of both biological molecules and complex ions by a candidatecompound. Drugs have been reported engage in redox reactions with bothcomplex ions and proteins. Ferric bleomycin, for example, has beenreported to catalyze the reduction of10-hydroperoxy-8,12-octadecadienoic acid in vitro. Padbury, G. et al.,Biochem. 27:7846-7852 (1988). Similarly, dopamine has been shown toundergo oxidation in the presence of L-cysteine. Shen, X. M. et al.,Chem. Res. Toxicol. 10:147-155 (1997). Further, beta-adrenergic receptorblocking medications are believed to oxidize proteins in vivo. Lamont,S., J. Cardpulm. Rehabil. 15:183-185 (1995).

[0068] A considerable number of assays are known in the art formeasuring redox reactions. For example, redox titrations may be used toidentify and quantify ionic species produced via redox reactions. Oneexample of such a titration involves the use of KMnO₄ which becomescolorless upon oxidizing Fe(II) to Fe(III). When all of the Fe(II) in asample has been converted to Fe(III), the titrated sample will retainthe purple color of the titration reagent. Brady, J. E. and Holum, J.R., FUNDAMENTALS OF CHEMISTRY, John Wiley and Sons, 1984, pages 392-396.Similar titration assays are also available for quantifying copper ionspecies. See, e.g., J. E. Brady and J. R. Holum, supra.

[0069] The present invention further encompasses methods for screeningcandidate compounds to identify those which induce TD by measuring theproduction of one or more intermediates or products of reactionsassociated with the reduction of reducible substrate or with theinduction of TD by antipsychotic drugs. These methods include, inaddition to redox assays, assays for detecting superoxide anions,hydrogen peroxide, and hydroxyl radicals generated by antipsychoticdrugs.

[0070] By the phrase “intermediates or products of reactions associatedwith the reduction of reducible substrate” is meant intermediates andend product directly or indirectly formed by reactions which occur as aresult of the reduction of reducible substrate (e.g., Fenton reactionproducts, hydrogen peroxide, superoxide anion).

[0071] A considerable number of assays are known for measuring both theconcentrations and production of the reactants and products shown inFIG. 11. Assays for detecting and quantifying superoxide anions as wellas other reactive oxygen species (e.g., H₂O₂ and OH^(•)), for example,are known in the art. See, e.g., Lemaire, P. and Livingston, D. R.,Comp. Biochem. Physiol. C. Pharmacol. Toxic. Endocrinol. 117:131-139(1997); Kelm, M. et al., J. Biol. Chem. 272:9922-9932 (1997); Masuoka,N. et al, Clin. Chim. Acta. 254:101-112 (1996); Zeller, J. M. andSullivan, B. L., J. Leukoc. Biol. 52:449-455 (1992). Such assays may bebased on a variety of different detection mechanisms includingcolorimetric detection (Lavoie, J. C. and Chessex, P., J. Pediatr.Gastroenterol. Nutr. 25:307-311 (1997)), the use of chemiluminescent(Kojima, S. et al., Anticancer Res. 14:1875-1880 (1995)) or fluorescentprobes (Biaglow, J. E. and Kachur, A. V., Radiat. Res. 148:181-187(1997)), and electron spin resonance spectroscopy (Lee, C. and Okabe,E., Jpn. J Pharmacol. 67:21-28 (1995)).

[0072] One embodiment of the present invention is shown in Example 1(b)where candidate compounds were tested for the ability to generatehydrogen peroxide in the presence of Cu(II). (FIGS. 3-5.) The presentinvention encompasses variations of the method disclosed in Example 1(b)where a reducible substrate is contacted with a candidate compound andthe generation of hydrogen peroxide is measured.

[0073] A second embodiment of the present invention is shown in Example1(c) where candidate compounds were tested for the ability to generatehydroxyl radicals in the presence of Cu(II). The present inventionencompasses variations of the method disclosed in Example 1(c) where areducible substrate is contacted with a candidate compound and thegeneration of hydroxyl radicals is measured.

[0074] As one skilled in the art would appreciate, once a candidatecompound has been identified by an assay of the present invention as onewhich does not induce TD, additional testing may be required to confirmthis finding. A number of method are available for confirming that thecandidate compound does not induce TD when administered to an animal.For example, multiple assays of the present invention may be performedusing the same candidate compound, after which, the data generated isused to predict whether the candidate compound will induce TD whenadministered to an animal. For instance, a candidate compo reductionactivity towards a reducible substrate but does not generate hydroxylradicals would not be predicted to induce TD.

[0075] The prediction that a candidate compound does not induce TD whenadministered to an animal can also be tested by assaying additionalcharacteristics of the compound. For example, a candidate compound whichhas reduction activity towards a reducible substrate, and has one ormore activities which eliminate reactive oxygen species would not beexpected to induce TD when administered to and animal. The antipsychoticdrug clozapine is such a compound and does not induce TD whenadministered to an animal. As noted above, the inventors have found thatclozapine may possess both superoxide dismutase and catalase activitieswhich would eliminate hydroxyl radical precursors prior to the formationof significant quantities of these radicals.

[0076] Confirmation of the prediction that a candidate compound does notinduce TD when administered to an animal can also be made using animalmodels. There are two major animal models of TD. The first is a primatemodel of antipsychotic-induced dyskinesia. Ten to thirty percents ofCebus or Maccaca monkeys will develop clinical dyskinesia afterlong-term antipsychotic treatment. The second is a rodent model ofvacuous chewing. In the rodent model, when the animals are treated withantipsychotics, they develop vacuous chewing, whic phenomenologicalsimilarities to the orobuccofacial movement of TD. Only 30-60% of ratswill develop vacuous chewing. The chewing movement is enhanced after thewithdrawal of antipsychotic treatment. Similar to the clinicalsituation, clozapine and vehicle treatment only induce vacuous chewingin a small portion of the animals. (FIG. 12.)

[0077] Animals which receive antipsychotic drugs and develop dyskinesiahave higher levels of reduced copper, hydrogen peroxide and hydroxyradical generation in the movement-related brain regions, such asstriatum. In vivo dialysis techniques can be applied to detect thechanges in redox active metals, as well as hydrogen peroxide and hydroxyradical generation. TD can be prevented or treated in the animals usedin this model by the chelation of copper and antioxidant administration.

[0078] Many of the assays useful in the practice of the presentinvention are readily adaptable for use in high-throughput assays forscreening candidate compounds to identify those which induce TD whenadministered to an animal. To achieve high-throughput screening, it isbest to house samples on a multicontainer carrier or platform. Amulticontainer carrier facilitates measuring reactions of a plurality ofcandidate compounds simultaneously. In one embodiment, a multi-wellmicroplate, for example a 96 or a 384 well microplate, which canaccommodate 96 or 384 different test reactions, is used as the carrier.Such multi-well microplates, and methods for their use in numerousassays, are both known in the art and commercially available. SigmaChemical Co., BIOCHEMICAL ORGANIC COMPOUND AND DIAGNOSTIC REAGENTS, 1996Catalog, pages 2134, 2483-2497.

[0079] In one embodiment, reactants are contained in each well of amulti-well microplate. The standard well(s) contains all of thereactants except the candidate compound. Each of the non-standard wellscontain at least one candidate compound. The reaction measured in thenon-standard wells are generally each compared against the reactionmeasured in the standard well(s).

[0080] Techniques for measuring the progression of reactions inmulticontainer carrier facilitates are known in the art and includespectrophotometry and spectrofluorometry. Colorimetric assays formeasuring the concentrations of chemical reactants are well known in theart. Examples of such assays are included in Example 1(a) and includethe use of bathocuproine disulfonate (BC) and bathophenanthrolinedisulfonate (BP) to measure the concentration of Cu(I) and Fe(II) ions,respectively. Further examples of such assays include the use ofcalorimetric assays for the detection of peroxides, as disclosed inExample 1(b) and Gordon, A. J. and Ford, R. A.,THE CHEMIST'S COMPANION:A HAND BOOK OF PRACTICAL DATA, TECHNIQUES, AND REFERENCES, John Wileyand Sons, N. Y., 1972, Page 437.

[0081] In one embodiment fluorescence spectrometry is used to monitorthe generation of reaction products. Fluorescence methodology isgenerally more sensitive than the absorption methodology. The use offluorescent probes is well known to those skilled in the art. Reviewedin Bashford, C. L. et al., SPECTROPHOTOMETRY AND SPECTROFLUOROMETRY: APRACTICAL APPROACH, pp.91-114, IRL Press Ltd. (1987); Bell, J. E.,SPECTROSCOPY IN BIOCHEMISTRY, Vol. 1, pp. 155-194, CRC Press (1981). Onemethod readily adaptable for use in high-throughput assays employs afluorescent probe, coumarin-3-carboxylic acid, for the detection ofhydroxyl radicals. Biaglow, J. E. and Kachur, A. V., Radiat Res.148:181-187 (1997).

[0082] Spectral readings can be taken on all of the samples housed in amulticontainer carrier simultaneously. Alternatively, readings can betaken on samples in groups of at least two at a time or one sample at atime. Further, the standard well(s) may be read at the same time as eachnon-standard well or the standard well(s) may be read prior orsubsequent to the reading of each non-standard well. The choice of whento read the samples containing the candidate compounds and the standardreaction mixture(s) will often vary with numerous factors includingwhether the reaction being assayed is continuously progressing or haseither reached an end point or has been “stopped” by other means (e.g.,via an alteration in pH or the addition of a chelating agent).

[0083] The present invention also provides methods for treatingpsychoses comprising the testing of candidate compounds to identifycompounds which will not induce tardive dyskinesia when administered toan animal, and administering one or more such compounds to a patient inneed thereof.

[0084] The antipsychotic drug(s) which will not induce TD isadministered to a patient as part of a pharmaceutically mixture atlevels sufficient to treat psychoses. The amount of the antipsychoticdrug administered to the patient will be formulated in accordance withmedical practice and will vary with such factors as the patient'scondition and the drug being administered.

[0085] The antipsychotic drug may be administered by any route thatdelivers efficacious levels of the drug, e.g., orally, intranasally,parenterally. For parenteral administration, preparations containing theantipsychotic drug may be provided to a patient in need of suchtreatment in combination with pharmaceutically acceptable sterileaqueous or non-aqueous solvents, suspensions and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oil, and injectable organic esters. Aqueous carriers includewater, water-alcohol solutions, Ringer's dextrose solution, dextroseplus sodium chloride solution, Ringer's solution containing lactose, orfixed oils.

[0086] Additional information related to dosages and the administrationof drugs can be found in numerous sources including REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition, 1990, Mack Publishing Co, Easton,Pa.

[0087] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.Examples 1(a) through 1(c), below, provide assays useful in the practiceof the present invention.

EXAMPLES Example 1

[0088] Screening assays for determining whether antipsychotics inducetardive dyskinesia

[0089] Three separate assays for determining whether candidateantipsychotic drugs induce TD when administered to animals are providedin this example.

Example 1(a)

[0090] Reduction of copper by antipsychotics

[0091] For the purpose of studying the ability of antipsychotics toreduce Cu(II) and Fe(III) under different conditions, we devised a novelhigh-throughput assay, based upon a modification of published protocols(Landers, J. W. and Zak, B., Clin. Chim. Acta. 3:328 (1958); Landers, J.W. and Zak, B., Amer. J. Clin. Path. 29:590-592 (1958); Sayre, L. M. andMulthaup, G., Science 274:1933-1934 (1996)), to operate in a 96-wellformat.

[0092] Method

[0093] To test whether antipsychotics possess metal reducing properties,10 ,μM of antipsychotics were co-incubated with 10 μM of Cu(II) andFe(III) in PBS (pH 7.4) at 37° C. for 1 hour. The Cu(I) and Fe(II)indicators, bathocuproine disulfonate (B C) and bathophenanthrolinedisulfonate (BP), respectively, were present at 200 μM during theincubation, and the formation of Cu(I)-BC and Fe(II)-BP complexes weremonitored at their absorption maxima: 483 nm and 536 nm, respectively,and concentrations calculated by comparison against standard curves. Theblanks were the metal solutions in PBS with their indicators without theaddition of antipsychotics. Therefore, the net production of reducedmetals by antipsychotics can be calculated based on the absorbancedifference between samples and blanks. The results indicate thatantipsychotics reduce Cu(II) more readily than Fe(III) (FIG. 1). Sincethe standard reduction potential for Cu(HI)/Cu(I) is much lower than Fe(III)/Fe(II), this result implies that reduction of Cu(II) may bespecifically favored by antipsychotics. Hence, antipsychotics canproduce highly reactive copper ion intermediates.

Example 1(b)

[0094] Generation of hydrogen peroxide by antipsychotics

[0095] To determine whether antipsychotics themselves could be thesource of peroxidation in TD, we measured hydrogen peroxide formation invitro using a novel 96-well assay developed specifically for thatpurpose. The production of hydrogen peroxide is assayed by amodification of a published method (Han, J. C. and Han, G. Y., Anal.Biochem. 220:5-10 (1994); Han, J., et al., Anal. Biochem. 234:107-109(1996)). We found that antipsychotics generate hydrogen peroxide in acopper-dependent manner. Antipsychotics (10 μM) were assayed forhydrogen peroxide formation in PBS (pH 7.4) in the presence of 10 μMCu(II). Approximately 5-15 μM hydrogen peroxide was formed byantipsychotics in one hour (FIG. 3). This signal (as with all hydrogenperoxide signals generated in these studies) was abolished by thepresence of catalase (100 U/ml), which rapidly removes hydrogenperoxide, confirming the validity of the assay.

[0096] Method

[0097] All buffers were treated with Chelex-100 to remove as much tracemetal contamination as possible, however this technique cannot removemetals to below the sub-picomolar level required to guarantee anenvironment that abolishes spontaneous dismutation, hence the presenceof a chelator is necessary to achieve such a baseline. Ten μM ofantipsychotics and 10 μM of Cu(II) were co-incubated with 100 μM of aH₂O₂-trapping reagent, Tris(2-Carboxyethyl)-Phosphine Hydrochloride(TCEP), in PBS (pH 7.4) at 37° C. for 1 hour. Then 100 μM of thedetection reagent, 5,5′-Dithio-bis(2-Nitrobenzoic Acid) (DTNB), wasadded to react with the remaining TCEP, the reaction product possessinga characteristic absorbance maximum at 412 nm. We adapted the hydrogenperoxide assay (Han, J., et al., Anal. Biochem. 234:107-109 (1996)) forthe rapid throughput study of antipsychotics by introducing a 96-wellformat, and by introducing a standard range of absorbance allowing us toempirically quantify hydrogen peroxide with a lower limit of detectionof 0.5 μM.

Example 1(c)

[0098] Generation of hydroxyl radical by antipsychotics

[0099] To determine whether antipsychotics themselves could be thesource of free radical generation in TD, we measured hydroxyl radicalformation in vitro using a novel 96-well assay developed specificallyfor that purpose. The method used to assay hydroxy radical is amodification of a published method (Gutteridge, J. M. C. and Wilkins,S., Biochim. Biophys. Acta 759:38-41 (1983)). Antipsychotics (10 μM)were assayed for hydroxyl radical formation based on the evidence thathydroxyl radical is capable of degrading deoxyribose (DOR) with theformation of thiobarbituric acid-reactive products. We found thatantipsychotics generate hydroxyl radical in a copper-dependent manner(FIG. 9).

[0100] Method

[0101] One μl of Cu(II) was added to 125 μl of 7.5 mM DOR and 10 μl of 1μM individual antipsychotic and made up to 500 μl with PBS (pH 7.4) in a1 ml tube. The reaction was incubated for 1 hour at 37° C. Afterincubation, 250 μl glacial acetic acid and 250 μl of thiobarbituric acid(1% w/v in 0.05 M NaOH) was added to the tubes which were then incubatedat 100° C. for 15-20 minutes. The absorbance was read at 532 mm. Weadapted the hydrogen peroxide assay for the rapid throughput study ofantipsychotics by introducing a 96-well formats (Han, J. C. and Han, G.Y., Anal. Biochem. 220:5-10 (1994); Han, J., et al., Anal. Biochem.234:107-109 (1996)).

[0102] Results

[0103] Reduction of metals

[0104] We found that conventional antipsychotics selectively reducecopper and, to a much less degree, iron. (FIG. 1.) On the other hand,while a few of the non-antipsychotic psychotropic drugs tested willreduce copper ions, most will not reduce significant quantities ofeither Cu(II) or Fe(III). (FIG. 2.)

[0105] Generation of hydrogen peroxide

[0106] Antipsychotics also generate hydrogen peroxide in addition totheir capacity to reduce metals. (FIG. 3.) On the other hand, manynon-antipsychotic psychotropics do no generate appreciable quantities ofhydrogen peroxide and those which do generate significantly hydrogenperoxide than most of the antipsychotics drugs tested. (FIG. 4.)

[0107] The capacity of the antipsychotic fluphenazine to generatehydrogen peroxide is concentration dependent, i.e. more fluphenazinegenerates more hydrogen peroxide. (FIG. 5.) The capacity of thisantipsychotic to generate hydrogen peroxide is also dependent on theconcentration of copper, i.e. more copper generates more hydrogenperoxide when reacted with antipsychotics. (FIG. 6.) When copper ischelated, the capacity of antipsychotics to generate hydrogen peroxideis significantly attenuated. (FIG. 7.)

[0108] In addition, the capacity of antipsychotics to generate hydrogenperoxide is dependent on oxygen, i.e. oxygen augments the production ofhydrogen peroxide by antipsychotics and argon, which expels oxygen inthe reaction, attenuates the production of hydrogen peroxide. (FIG. 8.)

[0109] Generation of hydroxyl radical

[0110] The reduction by antipsychotics of copper (II) to copper (I) maypromote an environment which enhances the production of hydroxylradicals and contributes to oxidative stress in TD. Consequently,conventional antipsychotics generate hydroxyl radicals by Fentonchemistry which may damage striatal neurons. (FIG. 9.) Therefore, braincopper homeostasis may be a variable that influences the ability ofantipsychotics to cause damage to striatal neurons. Copperconcentrations in the striatum are stringently regulated byenergy-dependent mechanisms, and mutations of a Cu-ATPase cause Wilson'sdisease, another neurodegenerative striatal disorder.

[0111] Interestingly, clozapine, antidepressants and other psychotropicsdid not generate hydroxyl radicals consistent with their low risk ofcausing TD. (FIGS. 9 and 10.) The inability of clozapine to generatehydroxy radicals in vitro may explain its lower propensity to produceTD.

[0112] We have found that clozapine may have superoxide dismutase andcatalase activities. Thus, clozapine appears to be atypical ofantipsychotic drugs in that, while it reduces Cu(II) ions (FIG. 1), thisdrug may also scavenge superoxide ions and hydrogen peroxide beforeappreciable quantities of hydroxyl radicals are formed.

[0113] Taken together, the ability of antipsychotics to produce hydrogenperoxide, and consequently hydroxyl radical, depend on the presence ofcopper and oxygen. The oxidative damage cascade is summarized in FIG.11. Our findings strongly suggest the redox activity of antipsychoticsmay underlie the pathogenesis of TD.

[0114] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0115] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0116] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

What is claimed is:
 1. A method for determining whether a candidatecompound induces tardive dyskinesia comprising: (a) contacting saidcandidate compound with a reducible substrate, (b) assaying for theproduction of one or more intermediates or products of reactionsassociated with the reduction of said reducible substrate, and (c)comparing the production of said intermediates or products to a standardproduction in a standard reaction mixture, wherein a significantincrease over the standard indicates that the candidate compound inducestardive dyskinesia when administered to an animal.
 2. The method ofclaim 1, wherein the reducible substrate has a standard reductionpotential between 0 and 2.0 E°/V.
 3. The method of claim 2, wherein thereducible substrate is a complex ion.
 4. The method of claim 2, whereinthe reducible substrate is Cu(II).
 5. The method of claim 1, wherein thereaction product assayed is hydrogen peroxide.
 6. The method of claim 1,wherein the reaction product assayed is hydroxyl radical.
 7. The methodof claim 1, wherein the reaction product assayed is superoxide anion. 8.The method of claim 1, wherein the reaction product assayed is reducedreducible substrate.
 9. The method of claim 1, wherein the ability ofthe candidate compound to induce tardive dyskinesia is confirmed byanimal testing.
 10. The method of claim 1, wherein the ability of thecandidate compound to induce tardive dyskinesia is confirmed byperforming assays for multiple intermediates and/or end products of thereactions which induce tardive dyskinesia.
 11. The method of claim 1,wherein assaying for reaction product(s) is performed using ahigh-throughput assay.
 12. The method of claim 11, wherein the reactionproduct is determined calorimetrically.
 13. The method of claim 11,wherein the reaction product is determined fluorometrically.
 14. Themethod of claim 11, wherein the assay is performed using samplescontained in a multicontainer carrier.
 15. The method of claim 11,wherein said candidate compound is an antipsychotic drug.
 16. A methodfor determining whether a candidate compound induces tardive dyskinesiacomprising: (a) incubating a candidate compound with a reduciblesubstrate, and (b) assaying for the production of a reactive oxygenspecies, whereby a determination of whether the reactive oxygen specieshas been produced is made by comparison to a standard reaction mixture,wherein a significant increase over the standard indicates that thecandidate compound induces tardive dyskinesia when administered to ananimal.
 17. The method of claim 16, wherein the reactive oxygen speciesis superoxide anion.
 18. The method of claim 16, wherein the reactiveoxygen species is hydroxyl radical.
 19. The method of claim 16, whereinthe reactive oxygen species is hydrogen peroxide.
 20. A methods fortreating an individual afflicted with a psychosis comprising: (a) thetesting of candidate compounds to identify those which will not inducetardive dyskinesia when administered to an animal, and (b) administeringone or more candidate compounds identified in (a) to a patient in needthereof.